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Figure 4 | Journal of Molecular Signaling

Figure 4

From: PNRC is a unique nuclear receptor coactivator that stimulates RNA polymerase III-dependent transcription

Figure 4

Down regulation of RNA Pol III gene transcription by specific depletion of endogenous PNRC using RNA interference. A, Northern blot analysis of PNRC and GAPDH mRNAs. MCF7 cells were transiently transfected with Pol III reporter plasmid, pArg maxi, along with 20 nM of either siRNA specific for PNRC (PNRC/si) or nonspecific mismatch RNA (mm). Twenty-four hours after transfection, 20 μg of total RNA, isolated from the transfected cells, was subjected to Northern analysis with PNRC and GAPDH cDNA probes, separately, as described in Methods section. B, Western blot of PNRC and actin protein. An aliquot of cell lysate equal to 100 μg of protein prepared from PNRC/siRNA (20 nM) transfected (PNRC/si) or nonspecific mismatch RNA (mm) transfected MCF7/EGFP-PNRC cells was separated on a 10% SDS-PAGE and subjected to Western analysis with 1:1000 diluted anti-GFP mouse monoclonal antibody (Cloetech) and anti-actin antibody (Santa Cruz Biotechnology) separately. The protein bands with molecular weights corresponding to those of EGFP-PNRC fusion protein or actin were indicated by arrows. C, Down regulation of tRNAarg transcription by PNRC/siRNA. MCF-7 cells were transiently transfected with reporter plasmid, pArg maxi (10 μg) along with 20 nM of either PNRC/siRNA (PNRC) or mismatch control RNA (mm). Twenty-four hours after transfection, the levels of tRNA transcripts in the transfected cells were determined by RNase protection assay as described in figure 3.

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